Optimising Umbilical Cord Mesenchymal Stem Cells for the Treatment of Keratoconus

Abstract

The cornea, a transparent ‘window’ of the eye, is composed of three distinct layers. Among these, keratocytes in the corneal stroma help maintain transparency, heal wounds, and synthesize stromal components. In Keratoconus, these cells are progressively lost such that the lamellar structure that maintains transparency is distorted, leading to vision loss. In serious instances, corneal transplants are performed; however, a global lack of donor tissue urges the need to develop alternative therapeutic strategies. The umbilical cord is an abundant source of mesenchymal stem cells (MSCs), which have the potential to differentiate into functional adult keratocytes. Yet, there are varying isolation and differentiation success rates. This research aimed to optimise the isolation of MSCs by exploring the characteristics and distribution of MSCs in the umbilical cord, and establishing the location with the largest number of MSCs. The structural and cellular anatomy of the umbilical cord in three dimensions were explored in umbilical cords by H&E and immunohistochemistry (IHC). A novel protocol for identifying MSCs in situ by IHC triple labelling of MSC markers was developed. Antibody validation was performed by Western Blot. Expression of these MSC markers were measured using droplet digital polymerase chain reaction. Cell distribution was quantified by DAPI staining. Vimentin, laminin, and αSMA were expressed by all the cells across cross-sections of sequential regions along the umbilical cord, confirming their mesenchymal origin, presence of basement membranes and myofibroblastic phenotypes. Cytokeratin was localised to the epithelial cord lining. All stromal cells in the umbilical cord displayed expression of CD105, CD90, and CD73, the three acclaimed positive MSC markers. CD34 and CD45, negative MSC markers, were also expressed. Cells furthest from the placental insertional region of the umbilical cord showed a statistically significant decrease in expression of CD105 and CD34, and increased CD90 expression. In terms of cell count, there were more cells surrounding the vasculature and at either ends of the umbilical cord than in the centre. These findings warrant further research into the functionality of MSC markers and the regions of the umbilical cord with the highest MSC potencies.